This proposal aims to obtain a detailed molecular understanding of how the gene for apolipoprotein AI (apoAI) is transcriptionally regulated in liver cells. ApoAI is the major protein component of high-density lipoprotein, HDL, whose plasma levels are inversely correlated with atherosclerosis, a leading cause of mortality and morbidity in developed countries. An aim of the project is also to understand the mechanism by which the orphan nuclear receptor HNF-4, previously implicated in apoAI transcriptional control, activates transcription by RNA polymerase II. In addition to apoAI, HNF-4 regulates many genes involved in the "metabolic syndrome" [including hypertension and non-insulin dependent diabetes mellitus (NIDDM)]. Also, its gene was independently isolated as the causative factor for maturity onset diabetes of the young (MODY1). Furthermore, HNF-4 is a major transcription factor for hepatitis B viral gene expression, an etiologic agent for liver cancer. The project is therefore expected to have broad relevance. Cellular and in vitro methods will be employed to identify transcription factors that are required for HNF-4-mediated activation at the liver-specific enhancer of the apoAI gene. Transcription factors under study will include another liver-enriched factor, HNF-3 (known to synergistically function with HNF-4), Mediator (the global coactivator recently shown to be necessary for HNF-4 function) and key chromatin cofactors (histone acetyl transferases, ATP-remodeling). Cellular analyses (chromatin immunoprecipitation [ChIP]) will delineate transcription factor interactions in living cells. Both steady-state expression of the apoAI gene in mature hepatocytes, as well as the pathway leading to the initial onset of apoAI gene expression in developing mouse embryoid bodies (in culture) will be monitored. In vitro approaches will complement these in vivo studies. Thus, functional (in vitro transcription from chromatin templates to recapitulate core features of apoAI gene regulation) and structural (protein-protein and protein-DNA interactions) analyses will also be undertaken. Together, the proposed studies will permit understanding of the mechanistic basis for the synergistic function of the relevant transcription factors, which regulate apoAI gene expression in vivo and contribute to its homeostasis. They will also potentially reveal targets for future therapeutic developments in the metabolic and cardiovascular areas. [unreadable] [unreadable]